Interferons (IFNs) are a group of cytokines that play essential roles in mediating innate immunity to a wide range of different types of viruses. The anti-viral activities of IFNs are due, in part, to RNA degradation by RNase L, a remarkable enzyme that is activated by 2',5' -oligoadenylates (2-5A). 2-5A is produced by a family of IFN-inducible synthetases that require double-stranded RNA (dsRNA), often of viral origin, for their activity. Interestingly, RNase L deficient mice, generated during this project, have enhanced susceptibility to lethal viral infections and are deficient in spontaneous and induced apoptosis. Our preliminary data further implicate 2-5A and RNase L as the mediators of a specific ribotoxic stress response that could result in the elimination of virus-infected cells. In this proposal, we aim to elucidate the specific role of the 2-5A/RNase L system in the overall biologic response to IFN. Our specific aims include to: (1) probe the function and regulation of RNase L by constructing chimeras between RNase L and the related nuclease Ire1; use two-hybrid systems and mass spectrometry to clone and identify host and vaccinia virus proteins that interact with and regulate RNase L; and perform structural studies on RNase L by NMR and crystallography; (2) determine how RNase L causes its anti-viral and pro-apoptotic activities by identifying the RNA substrate preferences of RNase L with DNA microarrays; investigate how RNase L contributes to dsRNA-activation of stress activated protein kinase JNK, and probe the apoptotic pathway initiated by 2-5A activation of RNase L; and (3) investigate the biologic actions of RNase L by studying involvement of RNase L in cellular immunity; determine the role of RNase L in resistance to HSV-1 infections, and dissect RNase L mediated responses to picomavirus infections in cultured primary cardiac myocytes and in organs of infected mice. RNase L is an important mediator of the anti-viral and apoptotic activities of IFNs. Activation of RNase L by a small and unique molecule, 2-5A, provides an opportunity to control viral infections at the level of RNA turnover. Therefore, continuation of this PROJECT is expected to lead to the eventual development of novel strategies for treatment of viral infections and cancer.